Polynucleotide ligases are enzymes which create a covalent bond between discontinuous nucleic acids. This joining of discontinuous nucleic acids by polynucleotide ligases plays a central role in a number of natural biological processes, including chromosome replication, genetic recombination and cellular repair of environmental genetic damage (e.g., X-ray damage). Polynucleotide ligases from virues, prokaryotic cells and eukaryotic cells have been described and characterized. In particular, NAD.sup.30 -dependent nucleic acid ligases of the eubacteria and the ATP-dependent nucleic acid ligases of eukaryotes are well characterized and serve essential functions in such cells.
Compounds which modulate ligase activity can act as cytotoxic agents by disrupting cellular processes in normal cell division processes. Modulators with adequate specificity can serve as antibacterial, antifungal, or antineoplastic agents. Similarly, as polynucleotide ligases are also virally encoded (or, in some cases, induced by viral infection), modulators of viral ligase activity can act as antiviral agents.
In addition to the role of polynucleotide ligases in biology and medicine, the ligation of polymerized nucleic acids is a necessary step in the fundamental techniques of molecular biology. Ligation of DNA and RNA polymers is ubiquitous in the common techniques of cloning, sequencing and analyzing genetic material. There are several general texts which describe ligation generally in recombinant techniques, such as Sambrook et al., Molecular Cloning--A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1997) ("Ausubel"). Similarly, techniques utilizing ligation are becoming increasingly important as general tools of basic research and in diagnostic clinical settings. The ligation chain reaction ("LCR"; sometimes denoted the "ligation amplification reaction" or "LAR") and related techniques are of increasing utility, particularly as diagnostic tools.
Given the many roles for ligase enzymes, ligase modulators which increase or decrease ligase activity, or which increase or decrease ligase specificity, would be useful in medicine, biology, or as general laboratory tools. Accordingly, various assays to identify compounds which affect ligase activity have been performed. For example, Tan et al. (1996) Biochem J. 314:993-1000 describe screening of potential inhibitors of human DNA ligase I (hLI) by mixing ligase reactants and plant extracts in microtiter dishes, followed by spotting the resulting products onto filter paper and scintillation counting.
Improved high-throughput assays which identify nucleic acid ligase modulators are, therefore, desirable. The present invention provides such high-throughput assays, as well as other features which will become apparent upon review.